Orca flash high speed camera

The effects of nanoMIL-89 on the cardiac function and development of the ZF embryos were assessed by imaging the heart and blood vessels at 72 hpf post embryo hatching. First, unhatched embryos were gently force hatched using forceps to unleash the larvae from their chorions. Then, the imaging of the heart muscle and blood flow video recordings were done for 5 embryos from each experimental group as previously described [34] (link). Briefly, ZF larvae were moved upon imaging into a depression glass slide in 3% methylcellulose, mixed with egg water to hinder their movement, and oriented in a lateral view, facing left with their yolk sac facing up. The imaging was done at a magnification of 100X for 5 s at 100 frames per second (fps) for the heart muscle contraction. Meanwhile, for the blood flow imaging, two major blood vessels, the dorsal aorta (DA), and posterior cardinal vein (PCV) were imaged at 100X for 10 s at 100 fps [34] (link). The video recordings were done using Zeiss SteREO Discovery V12 Microscope equipped with Hamamatsu Orca Flash high-speed camera and HCImage software V4.4.1.0 workstation (Hamamatsu Photonics, Japan).

Calcium flux studies were carried out by plating DIV120 astrocytes on glass bottom microwell dishes (MatTek, Germany) and allowed to reach 90% confluence. Next, cells were incubated in Tyrode’s solution (in mM: 129 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 25 HEPES, 30 Glucose, pH 7.4) supplemented with 5 μM Fluo-4 (ThermoFisher Scientific) and 0.02% Pluronic F-127 (Sigma) for 45 min at RT and 5% CO2 in the dark. Fluorescence microscopy was performed using an Olympus IX71 widefield microscope system (Olympus, Japan) with a ×20 objective, using time-series frames with an interval of 2 s, using Hamamatsu ORCA-Flash high speed camera (Hamamatsu Photonics, Belgium). For each biological replicate, 10–20 cells were measured. Traces in the graphs represent the normalized average fluorescence intensity change over time.